High sensitivity top-down proteomics captures single muscle cell heterogeneity in large proteoforms.

Single-cell proteomics has emerged as a powerful method to characterize cellular phenotypic heterogeneity and the cell-specific functional networks underlying biological processes. However, significant challenges remain in single-cell proteomics for the analysis of proteoforms arising from genetic mutations, alternative splicing, and post-translational modifications. Herein, we have developed a highly sensitive functionally integrated top-down proteomics method for the comprehensive analysis of proteoforms from single cells. We applied this method to single muscle fibers (SMFs) to resolve their heterogeneous functional and proteomic properties at the single-cell level. Notably, we have detected single-cell heterogeneity in large proteoforms (>200 kDa) from the SMFs. Using SMFs obtained from three functionally distinct muscles, we found fiber-to-fiber heterogeneity among the sarcomeric proteoforms which can be related to the functional heterogeneity. Importantly, we detected multiple isoforms of myosin heavy chain (~223 kDa), a motor protein that drives muscle contraction, with high reproducibility to enable the classification of individual fiber types. This study reveals single muscle cell heterogeneity in large proteoforms and establishes a direct relationship between sarcomeric proteoforms and muscle fiber types, highlighting the potential of top-down proteomics for uncovering the molecular underpinnings of cell-to-cell variation in complex systems.

Small-Scale Serial Size Exclusion Chromatography (s3SEC) for High Sensitivity Top-Down Proteomics of Large Proteoforms.

Top-down-mass spectrometry (MS)-based proteomics has emerged as a premier technology to examine proteins at the proteoform level, enabling characterization of genetic mutations, alternative splicing, and post-translational modifications. However, significant challenges that remain in top-down proteomics include the analysis of large proteoforms and the sensitivity required to examine proteoforms from minimal amounts of sample. To address these challenges, we have developed a new method termed "small-scale serial Size Exclusion Chromatography" (s3SEC), which incorporates a small-scale protein extraction (1 mg of tissue) and serial SEC without postfractionation sample handling, coupled with online high sensitivity capillary reversed-phase liquid chromatography tandem MS (RPLC-MS/MS) for analysis of large proteoforms. The s3SEC-RPLC-MS/MS method significantly enhanced the sensitivity and reduced the proteome complexity across the fractions, enabling the detection of high MW proteoforms previously undetected in one-dimensional (1D)-RPLC analysis. Importantly, we observed a drastic improvement in the signal intensity of high MW proteoforms in early fractions when using the s3SEC-RPLC method. Moreover, we demonstrate that this s3SEC-RPLC-MS/MS method also allows the analysis of lower MW proteoforms in subsequent fractions without significant alteration in proteoform abundance and equivalent or improved fragmentation efficiency to that of the 1D-RPLC approach. Although this study focuses on the use of cardiac tissue, the s3SEC-RPLC-MS/MS method could be broadly applicable to other systems with limited sample inputs.

ViaChip for Size-based Enrichment of Viable Cells.

Live cells acquire different fates including apoptosis, necrosis, and senescence in response to stress and stimuli. Rapid and label-free enrichment of live cells from a mixture of cells adopting various cell fates remains a challenge. We developed a ViaChip for high-throughput enrichment of Viable cells via size-based separation on a multi-stage microfluidic Chip. Our chip takes advantage of the characteristic increase in cell size during cellular senescence and decreases during apoptosis and necrosis, in comparison to their viable and healthy counterparts. The core component of our ViaChip is a slanted and tunable 3D filter array in the vertical direction (z-gap) for rapid and continuous cell sieving. The shape of the 3D filter array is optimized for target cells to prevent clogging during continuous separation. We demonstrated enrichment of live human and mouse mesenchymal stem cells in culture and from live animals, as well as the removal of senescent and necrotic MSCs, respectively, achieving an enrichment efficiency of ~67% with the continuous flow at 1.5 mL/hour. With further improvements in throughput and separation efficiency, our ViaChip could find applications in cell-based drug screening for anti-cancer and anti-aging cell therapies.

Multiplex Mass Spectrometry Analysis of Amyloid Proteins in Human Plasma for Alzheimer's Disease Diagnosis.

Direct analysis of amyloid proteins in human plasma will promote rapid screening of brain amyloidosis, the earliest pathological signature of Alzheimer's disease. We developed a microflow liquid chromatography-targeted mass spectrometry assay for quantitation of four intact ß-amyloid proteins starting from 1 mL of human plasma samples. This method showed 90% accuracy for predicting brain amyloid using plasma Aß42/Aß40 values from 36 cognitively normal individuals in a prospective clinical study (raw data deposited in MassIVE, Data set ID MSV000087451). Our method may contribute to early diagnosis of Alzheimer's disease.

Omics-Based Platform for Studying Chemical Toxicity Using Stem Cells.

The new strategy for chemical toxicity testing and modeling is to use in vitro human cell-based assays in conjunction with quantitative high-throughput screening (qHTS) technology, to identify molecular mechanisms and predict in vivo responses. Stem cells are more physiologically relevant than immortalized cell lines because of their unique proliferation and differentiation potentials. We established a robust two stem cells-two lineages assay system, encompassing human mesenchymal stem cells (hMSCs) along osteogenesis and human induced pluripotent stem cells (hiPSCs) along hepatogenesis. We performed qHTS phenotypic screening of LOPAC1280 and identified 38 preliminary hits for hMSCs. This was followed by validation of a selected number of hits and determination of their IC50 values and mechanistic studies of idarubicin and cantharidin treatments using proteomics and bioinformatics. In general, hiPSCs were more sensitive than hMSCs to chemicals, and differentiated progenies were less sensitive than their progenitors. We showed that chemical toxicity depends on both stem cell types and their differentiation stages. Proteomics identified and quantified over 3000 proteins for both stem cells. Bioinformatics identified apoptosis and G2/M as the top pathways conferring idarubicin toxicity. Our Omics-based assays of stem cells provide mechanistic insights into chemical toxicity and may help prioritize chemicals for in-depth toxicological evaluations.

Quantitation of Intact Proteins in Human Plasma Using Top-Down Parallel Reaction Monitoring-MS.

Direct quantitation of proteins in complex biological matrices by mass spectrometry remains a challenge. Here, we describe a novel top-down parallel reaction monitoring-mass spectrometry (PRM-MS) assay, enabled by microflow LC-nanospray MS using a silicon microfluidic LC-MS chip. We demonstrated direct analysis of intact proteins such as somatropin in human plasma, achieving sensitivity (0.1-1.0 fmole) and speed (1-5 min) on par with enzyme-linked immunosorbent assay (ELISA).

14-3-3σ stabilizes a complex of soluble actin and intermediate filament to enable breast tumor invasion.

The protein 14-3-3σ (stratifin) is frequently described as a tumor suppressor silenced in about 80% of breast tumors. Intriguingly, we show that 14-3-3σ expression, which in normal breast is localized to the myoepithelial cells, tracks with malignant phenotype in two models of basal-like breast cancer progression, and in patients, it is associated with basal-like subtype and poor clinical outcome. We characterized a mechanism by which 14-3-3σ guides breast tumor invasion by integrating cytoskeletal dynamics: it stabilizes a complex of solubilized actin and intermediate filaments to maintain a pool of "bioavailable" complexes for polarized assembly during migration. We show that formation of the actin/cytokeratin/14-3-3σ complex and cellular migration are regulated by PKCζ-dependent phosphorylation, a finding that could form the basis for intervention in aggressive breast carcinomas expressing 14-3-3σ. Our data suggest that the biology of this protein is important in cellular movement and is contingent on breast cancer subtype.

Biomonitoring of perfluorinated compounds in a drop of blood.

Biomonitoring of pollutants and their metabolites and derivatives using biofluids provides new opportunities for spatiotemporal assessment of human risks to environmental exposures. Perfluorinated compounds (PFCs) have been used widely in industry and pose significant environmental concerns due to their stability and bioaccumulation in humans and animals. However, current methods for extraction and measurement of PFCs require relatively large volumes (over one hundred microliters) of blood samples, and therefore, are not suitable for frequent blood sampling and longitudinal biomonitoring of PFCs. We have developed a new microassay, enabled by our silicon microfluidic chip platform, for analyzing PFCs in small volumes (less than five microliters) of blood. Our assay integrates on-chip solid-phase extraction (SPE) with online nanoflow liquid chromatography-electrospray ionization-mass spectrometry (nanoLC-ESI-MS) detection. We demonstrated high sample recovery, excellent interday and intraday accuracy and precision, and a limit of detection down to 50 femtogram of PFCs, in one microliter of human plasma. We validated our assay performance using pooled human plasma and NIST SRM 1950 samples. Our microfluidic chip-based assay may enable frequent longitudinal biomonitoring of PFCs and other environmental toxins using a finger prick of blood, thereby providing new insights into their bioaccumulation, bioavailability, and toxicity.

Top-down proteomics of a drop of blood for diabetes monitoring.

The most common markers for monitoring patients with diabetes are glucose and HbA1c, but additional markers such as glycated human serum albumin (HSA) have been identified that could address the glycation gap and bridge the time scales of glycemia between transient and 2-3 months. However, there is currently no technical platform that could measure these markers concurrently in a cost-effective manner. We have developed a new assay that is able to measure glucose, HbA1c, glycated HSA, and glycated apolipoprotein A-I (apoA-I) for monitoring of individual blood glycemia, as well as cysteinylated HSA, S-nitrosylated HbA, and methionine-oxidized apoA-I for gauging oxidative stress and cardiovascular risks, all in 5 µL of blood. The assay utilizes our proprietary multinozzle emitter array chip technology to enable the analysis of small volumes of blood, without complex sample preparation prior to the online and on-chip liquid chromatography-nanoelectrospray ionization mass spectrometry. Importantly, the assay employs top-down proteomics for more accurate quantitation of protein levels and for identification of post-translational modifications. Further, the assay provides multimarker, multitime-scale, and multicompartment monitoring of blood glycemia. Our assay readily segregates healthy controls from Type 2 diabetes patients and may have the potential to enable better long-term monitoring and disease management of diabetes.

RNA helicase DDX5 regulates microRNA expression and contributes to cytoskeletal reorganization in basal breast cancer cells.

RNA helicase DDX5 (also p68) is involved in all aspects of RNA metabolism and serves as a transcriptional coregulator, but its functional role in breast cancer remains elusive. Here, we report an integrative biology study of DDX5 in breast cancer, encompassing quantitative proteomics, global MicroRNA profiling, and detailed biochemical characterization of cell lines and human tissues. We showed that protein expression of DDX5 increased progressively from the luminal to basal breast cancer cell lines, and correlated positively with that of CD44 in the basal subtypes. Through immunohistochemistry analyses of tissue microarrays containing over 200 invasive human ductal carcinomas, we observed that DDX5 was up-regulated in the majority of malignant tissues, and its expression correlated strongly with those of Ki67 and EGFR in the triple-negative tumors. We demonstrated that DDX5 regulated a subset of MicroRNAs including miR-21 and miR-182 in basal breast cancer cells. Knockdown of DDX5 resulted in reorganization of actin cytoskeleton and reduction of cellular proliferation. The effects were accompanied by up-regulation of tumor suppressor PDCD4 (a known miR-21 target); as well as up-regulation of cofilin and profilin, two key proteins involved in actin polymerization and cytoskeleton maintenance, as a consequence of miR-182 down-regulation. Treatment with miR-182 inhibitors resulted in morphologic phenotypes resembling those induced by DDX5 knockdown. Using bioinformatics tools for pathway and network analyses, we confirmed that the network for regulation of actin cytoskeleton was predominantly enriched for the predicted downstream targets of miR-182. Our results reveal a new functional role of DDX5 in breast cancer via the DDX5→miR-182→actin cytoskeleton pathway, and suggest the potential clinical utility of DDX5 and its downstream MicroRNAs in the theranostics of breast cancer.

Multinozzle emitter array chips for small-volume proteomics.

High-throughput multiplexed proteomics of small-volume biospecimens will generate new opportunities in theranostics. Achieving parallel top-down and bottom-up mass spectrometry analyses of target proteins using a unified apparatus will improve proteome characterization. We have developed a novel silicon-based microfluidic device, multinozzle emitter array chip (MEA chip), as a new platform for small-volume proteomics using liquid chromatography-nanoelectrospray ionization mass spectrometry (LC-nanoESI-MS). We demonstrate parallel, on-chip, and online LC-MS analysis of hemoglobin and its tryptic digests directly from microliters of blood, achieving a detection limit of less than 5 red blood cells. Our MEA chip will enable clinical proteomics of small-volume samples.

Multinozzle emitter arrays for nanoelectrospray mass spectrometry.

Mass spectrometry (MS) is the enabling technology for proteomics and metabolomics. However, dramatic improvements in both sensitivity and throughput are still required to achieve routine MS-based single cell proteomics and metabolomics. Here, we report the silicon-based monolithic multinozzle emitter array (MEA) and demonstrate its proof-of-principle applications in high-sensitivity and high-throughput nanoelectrospray mass spectrometry. Our MEA consists of 96 identical 10-nozzle emitters in a circular array on a 3 in. silicon chip. The geometry and configuration of the emitters, the dimension and number of the nozzles, and the micropillar arrays embedded in the main channel can be systematically and precisely controlled during the microfabrication process. Combining electrostatic simulation and experimental testing, we demonstrated that sharpened-end geometry at the stem of the individual multinozzle emitter significantly enhanced the electric fields at its protruding nozzle tips, enabling sequential nanoelectrospray for the high-density emitter array. We showed that electrospray current of the multinozzle emitter at a given total flow rate was approximately proportional to the square root of the number of its spraying-nozzles, suggesting the capability of high MS sensitivity for multinozzle emitters. Using a conventional Z-spray mass spectrometer, we demonstrated reproducible MS detection of peptides and proteins for serial MEA emitters, achieving sensitivity and stability comparable to the commercial capillary emitters. Our robust silicon-based MEA chip opens up the possibility of a fully integrated microfluidic system for ultrahigh-sensitivity and ultrahigh-throughput proteomics and metabolomics.

Comparative ultrastructure of trichomes on various organs of Rosa roxburghii.

Chestnut rose, R. roxburghii Tratt. (Rosaceae) (RR) is an important crop in China due to its nutritional and medicinal values. RR frequently produces trichomes on the surfaces of a diverse range of organs, however a genetic component exists to the control of trichome development, with some cultivars having significantly fewer trichomes to others. Certain varieties have fruits that are thickly covered with macroscopic trichomes, which is an undesirable trait for fruit processing and consumption. However, smooth-fruit cultivars exist, such as R. roxburghii Tratt. f. esetosa Ku (RRE). Despite their economic importance, the anatomical features of trichomes have not been explored in detail for these two chestnut rose germplasms. Here, we investigate the ultrastructure of trichomes distributed on the stem, sepal, and fruit of RR and RRE using transmission electron microscopy (TEM). The internal structure of stem prickle trichomes in RR and RRE was oval in shape and did not contain nucleoli or other organelles. The cell walls of stem prickles in RR are thick and the intercellular spaces occupied with liquid, whereas the cells wall of stem prickles in RRE are thin and have air-filled intercellular spaces. The cells of sepal acicular trichomes in RR and glandular trichomes (GTs) of sepals in RRE had similar vacuole sizes, cytoplasm content, intercellular spaces, and arrangement of plastids within cells. However, there were osmiophilic granules present in the GTs of RRE. The flagelliform trichomes in the sepals of the two germplasms are composed of oval or rod-shaped cells. Although the flagelliform trichomes in the sepals of the two germplasms had a similar internal structure, and both contained starch grains and plastids with visible thylakoid membranes, the flagelliform trichomes in the sepals of RR had a thinner cell wall and a higher proportion of cytoplasm which was more evenly distributed across the cell. There were granules that stained heavily with osmium tetroxide which occurred infrequently in the flagelliform trichomes of sepals in RRE but were not observed in RR. On the acicular trichomes of fruit in RR, the flagelliform trichomes and the GTs of fruit in RRE shared similar cell morphology, arrangement and vacuole size as well as intercellular space. Both the fruit flagelliform trichomes and GTs in RRE contain granules which stain heavily with osmium tetroxide, and the GTs contain plastids and starch grains. These differences in trichome cell ultrastructure may be related to developmental processes or biological functions of the trichomes. These results also suggest that the two chestnut rose germplasms are good candidates for further study of trichome ontogeny in the genus and subsequent breeding of the smooth organ trait in this species.

Senescence chips for ultrahigh-throughput isolation and removal of senescent cells.

Cellular senescence plays an important role in organismal aging and age-related diseases. However, it is challenging to isolate low numbers of senescent cells from small volumes of biofluids for downstream analysis. Furthermore, there is no technology that could selectively remove senescent cells in a high-throughput manner. In this work, we developed a novel microfluidic chip platform, termed senescence chip, for ultrahigh-throughput isolation and removal of senescent cells. The core component of our senescence chip is a slanted and tunable 3D micropillar array with a variety of shutters in the vertical direction for rapid cell sieving, taking advantage of the characteristic cell size increase during cellular senescence. The 3D configuration achieves high throughput, high recovery rate, and device robustness with minimum clogging. We demonstrated proof-of-principle applications in isolation and enumeration of senescent mesenchymal stem cells (MSCs) from undiluted human whole blood, and senescent cells from mouse bone marrow after total body irradiation, with the single-cell resolution. After scale-up to a multilayer and multichannel structure, our senescence chip achieved ultrahigh-throughput removal of senescent cells from human whole blood with an efficiency of over 70% at a flow rate of 300 ml/hr. Sensitivity and specificity of our senescence chips could be augmented with implementation of multiscale size separation, and identification of background white blood cells using their cell surface markers such as CD45. With the advantages of high throughput, robustness, and simplicity, our senescence chips may find wide applications and contribute to diagnosis and therapeutic targeting of cellular senescence.

Proteoform-Specific Protein Binding of Small Molecules in Complex Matrices.

Characterizing the specific binding between protein targets and small molecules is critically important for drug discovery. Conventional assays require isolation and purification of small molecules from complex matrices through multistep chromatographic fractionation, which may alter their original bioactivity. Most proteins undergo posttranslational modification, and only certain proteoforms have the right conformation with accessible domains and available residues for small molecule binding. We developed a top-down mass spectrometry (MS) centric workflow for rapid evaluation of the bioactivity of crude botanical extracts after a one-step reaction. Our assay distinguished covalent from noncovalent binding and mapped the residue for covalent binding between bioactive constituents and specific proteoforms of the target protein. We augmented our approach with a nanoflow liquid chromatography-selected reaction monitoring (SRM)-MS assay for simultaneous identification and label-free multiplex quantitation of small molecules in the crude botanical extracts. Our assay was validated for various proteoforms of human serum albumin, which plays a key role in pharmacokinetics of small molecules in vivo. We demonstrated the utility of our proteoform-specific assay for evaluating thymoquinone in crude botanical extracts, studying its pharmacokinetics in human blood, and interpreting its toxicity to human breast cancer cells in tissue culture.

Site mutations disrupt inter-helical H-bonds (alpha14W-alpha67T and beta15W-beta72S) involved in kinetic steps in the hemoglobin R-->T transition without altering the free energies of oxygenation.

Three recombinant mutant hemoglobins (rHbs) of human normal adult hemoglobin (Hb A), rHb (alphaT67V), rHb (betaS72A), and rHb (alphaT67V, betaS72A), have been constructed to test the role of the tertiary intra-subunit H-bonds between alpha67T and alpha14W and between beta72S and beta15W in the cooperative oxygenation of Hb A. Oxygen-binding studies in 0.1 M sodium phosphate buffer at 29 degrees C show that rHb (alphaT67V), rHb (betaS72A), and rHb (alphaT67V, betaS72A) exhibit oxygen-binding properties similar to those of Hb A. The binding of oxygen to these rHbs is highly cooperative, with a Hill coefficient of approximately 2.8, compared to approximately 3.1 for Hb A. Proton nuclear magnetic resonance (NMR) studies show that rHb (alphaT67V), rHb (betaS72A), rHb (alphaT67V, betaS72A), and Hb A have similar quaternary structures in the alpha(1)beta(2) subunit interfaces. In particular, the inter-subunit H-bonds between alpha42Tyr and beta99Asp and between beta37Trp and alpha94Asp are maintained in the mutants in the deoxy form. There are slight perturbations in the distal heme pocket region of the alpha- and beta-chains in the mutants. A comparison of the exchangeable 1H resonances of Hb A with those of these three rHbs suggests that alpha67T and beta72S are H-bonded to alpha14W and beta15W, respectively, in the CO and deoxy forms of Hb A. The absence of significant free energy changes for the oxygenation process of these three rHbs compared to those of Hb A, even though the inter-helical H-bonds are abolished, indicates that these two sets of H-bonds are of comparable strength in the ligated and unligated forms of Hb A. Thus, the mutations at alphaT67V and betaS72A do not affect the overall energetics of the oxygenation process. The preserved cooperativity in the binding of oxygen to these three mutants also implies that there are multiple interactions involved in the oxygenation process of Hb A.

Mode recognition in UV resonance Raman spectra of imidazole: histidine monitoring in proteins.

The imidazole side-chains of histidine residues perform key roles in proteins, and spectroscopic markers are of great interest. The imidazole Raman spectrum is subject to resonance enhancement at UV wavelengths, and a number of UVRR markers of structure have been investigated. We report a systematic experimental and computational study of imidazole UVRR spectra, which elucidates the band pattern, and the effects of protonation and deprotonation, of H/D exchange, of metal complexation, and of addition of a methyl substituent, modeling histidine itself. A consistent assignment scheme is proposed, which permits tracking of the bands through these chemical variations. The intensities are dominated by normal mode contributions from stretching of the strongest ring bonds, C(2)N and C(4)C(5), consistent with enhancement via resonance with a dominant imidazole π-π* transition.

Single cell analysis: the new frontier in 'omics'.

Cellular heterogeneity that arises from stochastic expression of genes, proteins and metabolites is a fundamental principle of cell biology, but single cell analysis has been beyond the capability of 'omics' technology. This is rapidly changing with the recent examples of single cell genomics, transcriptomics, proteomics and metabolomics. The rate of change is expected to accelerate owing to emerging technologies that range from micro/nanofluidics to microfabricated interfaces for mass spectrometry to third- and fourth-generation automated DNA sequencers. As described in this review, single cell analysis is the new frontier in omics, and single cell omics has the potential to transform systems biology through new discoveries derived from cellular heterogeneity.